Mesenchymal Stem Cell-Derived Mitochondria Enhance Extracellular Matrix-Derived Grafts for the Repair of Nerve Defect.

Peripheral nerve injuries (PNI) can lead to mitochondrial dysfunction and energy depletion within the affected microenvironment. The objective is to investigate the potential of transplanting mitochondria to reshape the neural regeneration microenvironment. High-purity functional mitochondria with an intact structure are extracted from human umbilical cord-derived mesenchymal stem cells (hUCMSCs) using the Dounce homogenization combined with ultracentrifugation. Results show that when hUCMSC-derived mitochondria (hUCMSC-Mitos) are cocultured with Schwann cells (SCs), they promote the proliferation, migration, and respiratory capacity of SCs. Acellular nerve allografts (ANAs) have shown promise in nerve regeneration, however, their therapeutic effect is not satisfactory enough. The incorporation of hUCMSC-Mitos within ANAs has the potential to remodel the regenerative microenvironment. This approach demonstrates satisfactory outcomes in terms of tissue regeneration and functional recovery. Particularly, the use of metabolomics and bioenergetic profiling is used for the first time to analyze the energy metabolism microenvironment after PNI. This remodeling occurs through the enhancement of the tricarboxylic acid cycle and the regulation of associated metabolites, resulting in increased energy synthesis. Overall, the hUCMSC-Mito-loaded ANAs exhibit high functionality to promote nerve regeneration, providing a novel regenerative strategy based on improving energy metabolism for neural repair.

Light-Driven Electron Uptake from Nonfermentative Organic Matter to Expedite Nitrogen Dissimilation by Chemolithotrophic Anammox Consortia.

Nonphotosynthetic microorganisms are typically unable to directly utilize light energy, but light might change the metabolic pathway of these bacteria indirectly by forming intermediates such as reactive oxygen species (ROS). This work investigated the role of light on nitrogen conversion by anaerobic ammonium oxidation (anammox) consortia. The results showed that high intensity light (>20000 lx) caused ca. 50% inhibition of anammox activity, and total ROS reached 167% at 60,000 lx. Surprisingly, 200 lx light was found to induce unexpected promotion of the nitrogen conversion rate, and ultraviolet light (<420 nm) was identified as the main contributor. Metagenomic and metatranscriptomic analyses revealed that the gene encoding cytochrome c peroxidase was highly expressed only under 200 lx light. 15N isotope tracing, gene abundance quantification, and external H2O2 addition experiments showed that photoinduced trace H2O2 triggered cytochrome c peroxidase expression to take up electrons from extracellular nonfermentative organics to synthesize NADH and ATP, thereby expediting nitrogen dissimulation of anammox consortia. External supplying reduced humic acid into a low-intensity light exposure system would result in a maximal 1.7-fold increase in the nitrogen conversion rate. These interesting findings may provide insight into the niche differentiation and widespread nature of anammox bacteria in natural ecotopes.

Dual-bionic regenerative microenvironment for peripheral nerve repair.

Autologous nerve grafting serves is considered the gold standard treatment for peripheral nerve defects; however, limited availability and donor area destruction restrict its widespread clinical application. Although the performance of allogeneic decellularized nerve implants has been explored, challenges such as insufficient human donors have been a major drawback to its clinical use. Tissue-engineered neural regeneration materials have been developed over the years, and researchers have explored strategies to mimic the peripheral neural microenvironment during the design of nerve catheter grafts, namely the extracellular matrix (ECM), which includes mechanical, physical, and biochemical signals that support nerve regeneration. In this study, polycaprolactone/silk fibroin (PCL/SF)-aligned electrospun material was modified with ECM derived from human umbilical cord mesenchymal stem cells (hUMSCs), and a dual-bionic nerve regeneration material was successfully fabricated. The results indicated that the developed biomimetic material had excellent biological properties, providing sufficient anchorage for Schwann cells and subsequent axon regeneration and angiogenesis processes. Moreover, the dual-bionic material exerted a similar effect to that of autologous nerve transplantation in bridging peripheral nerve defects in rats. In conclusion, this study provides a new concept for designing neural regeneration materials, and the prepared dual-bionic repair materials have excellent auxiliary regenerative ability and further preclinical testing is warranted to evaluate its clinical application potential.

Electrical stimulation accelerates Wallerian degeneration and promotes nerve regeneration after sciatic nerve injury.

Following peripheral nerve injury (PNI), Wallerian degeneration (WD) in the distal stump can generate a microenvironment favorable for nerve regeneration. Brief low-frequency electrical stimulation (ES) is an effective treatment for PNI, but the mechanism underlying its effect on WD remains unclear. Therefore, we hypothesized that ES could enhance nerve regeneration by accelerating WD. To verify this hypothesis, we used a rat model of sciatic nerve transection and provided ES at the distal stump of the injured nerve. The injured nerve was then evaluated after 1, 4, 7, 14 and 21 days post injury (dpi). The results showed that ES significantly promoted the degeneration and clearance of axons and myelin, and the dedifferentiation of Schwann cells. It upregulated the expression of BDNF and NGF and increased the number of monocytes and macrophages. Through transcriptome sequencing, we systematically investigated the effect of ES on the molecular processes involved in WD at 4 dpi. Evaluation of nerves bridged using silicone tubing after transection showed that ES accelerated early axonal and vascular regeneration while delaying gastrocnemius atrophy. These results demonstrate that ES promotes nerve regeneration by accelerating WD and upregulating the expression of neurotrophic factors.

A review of anammox-based nitrogen removal technology: From microbial diversity to engineering applications.

The anaerobic ammonium oxidation (anammox) process has the advantages of high efficiency and low energy consumption, so it has broad application prospects in biological denitrification of wastewater. However, the application of anammox technology to existing wastewater treatment is still challenging. The main problems are the insufficient supply of nitrite and the susceptibility of anammox bacteria to environmental factors. In this paper, from the perspective of the diversity of anammox bacteria, the habitats and characteristics of anammox bacteria of different genera were compared. At the same time, laboratory research and engineering applications of anammox technology in treating wastewater from different sources were reviewed, and the progress of and obstacles to the practical application of anammox technology were clarified. Finally, a focus for future research was proposed to intensively study the water quality barrier factors of anammox and its regulation strategies. Meanwhile, a combined process was developed and optimized on this basis.

Inorganic quantum dots - anammox consortia hybrid for stable nitrogen elimination under high-intensity solar-simulated irradiation.

External stimulus such as light irradiation is able to deteriorate intracellular redox homeostasis and induce photooxidative damage to non-photogenic bacteria. Exploiting effective strategies to help bacteria resisting infaust stress is meaningful for achieving a stable operation of biological treatment system. In this work, selenium-doped carbon quantum dots (Se-CQDs) were blended into anaerobic ammonia oxidation (anammox) bacteria and an inorganic nanoparticle-microbe hybrid was successfully fabricated to evaluate its nitrogen removal performance under solar-simulated irradiation. It was found that the specific anammox activity decreased by 29.7 ± 5.2% and reactive oxygen species (ROS) content increased by 134.8 ± 4.1% under 50,000 lux light. Sludge activity could be completely recovered under the optimum dosage of 0.42 mL·(g volatile suspended solid) -1 Se-CQDs. Hydroxyl radical (·OH) and superoxide anion radical (·O2-) were identified as the leading ROS inducing lipid peroxidation and antioxidase function detriment. Also, the structure of ladderane lipids located on anammoxosome was destroyed by ROS and functional genes abundances declined accordingly. Although cell surface coated Se-CQDs could absorb ultraviolet light and partially mitigated the photoinhibition, the direct scavenging of ROS by intracellular Se-CQDs primarily contributed to the cellular redox homeostasis, antioxidase activity recovery and sludge activity improvement. The findings of this work provide in-depth understanding the metabolic response mechanism of anammox consortia to light irradiation and might be valuable for a more stable and sustainable nitrogen removal technology, i.e., algal-bacterial symbiotic system, development.

Molecular spectroscopy and docking simulation revealed the binding mechanism of phenol onto anammox sludge extracellular polymeric substances.

The rapid development of chemical industry has induced to the large amount of phenolic wastewater production. When the promising anaerobic ammonium oxidation (anammox) was employed to treat the industrial wastewater, phenolic compounds would possibly inhibit the microbial performance. Extracellular polymeric substances (EPSs) play an essential role in protecting cells from being intoxicated by phenolic compound while the distinct mechanism remains elusive. In this work, the interaction of phenol with anammox sludge EPSs and transmembrane ammonium transport (Amt) domain was explored at molecular level by using spectral method and molecular docking simulation. It was found that phenol statically quenched the fluorescent components of EPSs and the protein component dominated the interaction between EPSs and phenol. The overall interaction was an entropy-driven process with hydrophobic interaction as the main driving force, and the CO vibration responded preferentially. As phenol continued to penetrate into the cell surface, there were hydrogen bond, hydrophobic interaction force and π-π base-stacking forces between the Amt domain and phenol. The interaction between phenol and amino acid residues of the Amt domain would interfere the NH4+ transport and further affect the activity of anammox sludge. This work is beneficial for in-depth understanding the role of EPSs in protecting anammox sludge from inhibiting by phenolic pollutants.

Functional tissue-engineered microtissue formed by self-aggregation of cells for peripheral nerve regeneration.

BACKGROUND: Peripheral nerve injury (PNI) is one of the essential causes of physical disability with a high incidence rate. The traditional tissue engineering strategy, Top-Down strategy, has some limitations. A new tissue-engineered strategy, Bottom-Up strategy (tissue-engineered microtissue strategy), has emerged and made significant research progress in recent years. However, to the best of our knowledge, microtissues are rarely used in neural tissue engineering; thus, we intended to use microtissues to repair PNI. METHODS: We used a low-adhesion cell culture plate to construct adipose-derived mesenchymal stem cells (ASCs) into microtissues in vitro, explored the physicochemical properties and microtissues components, compared the expression of cytokines related to nerve regeneration between microtissues and the same amount of two-dimension (2D)-cultured cells, co-cultured directly microtissues with dorsal root ganglion (DRG) or Schwann cells (SCs) to observe the interaction between them using immunocytochemistry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA). We used grafts constructed by microtissues and polycaprolactone (PCL) nerve conduit to repair sciatic nerve defects in rats. RESULTS: The present study results indicated that compared with the same number of 2D-cultured cells, microtissue could secrete more nerve regeneration related cytokines to promote SCs proliferation and axons growth. Moreover, in the direct co-culture system of microtissue and DRG or SCs, axons of DRG grown in the direction of microtissue, and there seems to be a cytoplasmic exchange between SCs and ASCs around microtissue. Furthermore, microtissues could repair sciatic nerve defects in rat models more effectively than traditional 2D-cultured ASCs. CONCLUSION: Tissue-engineered microtissue is an effective strategy for stem cell culture and therapy in nerve tissue engineering.

A review on characterizing the metabolite property of anammox sludge by spectroscopy.

As one of the most promising autotrophic biological nitrogen removal technology, anaerobic ammonia oxidation (anammox) has gained intense attention for the past decades and several full-scale facilities have been implemented worldwide. However, anammox bacteria are easily affected by disturbed external environmental factors, which commonly leads to the fluctuations in reactor performance. The response of anammox sludge to external stress results in changes in components and structural characteristics of intracellular and extracellular polymer substances. Real-time and convenient spectral analysis of anammox sludge metabolites can give early warning of performance deterioration under external stresses, which is of great significance to the stable operation of bioreactor. This review summarized the research progress on characterizing the intracellular and extracellular metabolites of anammox sludge through spectroscopic techniques. The correlation between anammox sludge activity and its key metabolites was analyzed. Also, the limitations and future prospects of applying spectral analytical techniques for anammox bioreactor monitoring were discussed and outlooked. This review may provide valuable information for both scientific study and engineering application of anammox based nitrogen removal technology.

Cell-Derived Extracellular Matrix Materials for Tissue Engineering.

The involvement of cell-derived extracellular matrix (CDM) in assembling tissue engineering scaffolds has yielded significant results. CDM possesses excellent characteristics, such as ideal cellular microenvironment mimicry and good biocompatibility, which make it a popular research direction in the field of bionanomaterials. CDM has significant advantages as an expansion culture substrate for stem cells, including stabilization of phenotype, reversal of senescence, and guidance of specific differentiation. In addition, the applications of CDM-assembled tissue engineering scaffolds for disease simulation and tissue organ repair are comprehensively summarized; the focus is mainly on bone and cartilage repair, skin defect or wound healing, engineered blood vessels, peripheral nerves, and periodontal tissue repair. We consider CDM as a highly promising bionic biomaterial for tissue engineering applications and propose a vision for its comprehensive development. Impact statement Cell-derived extracellular matrix (CDM) has received continued attention on the field of tissue engineering because of its promising biological characteristics. CDM deposited in vitro is rich in protein fractions and contains a wealth of biological information that provides a suitable niche for the survival and activity of isolated cells. More importantly, the free-assembling feature of CDM allows it to participate in the assembly of tissue-engineered scaffolds, imparting bionic properties to regenerative scaffolds, and thus CDM-modified scaffolds are widely used in the reconstruction of bone and cartilage tissue, peripheral nerves, skin, and blood vessels. This article is dedicated to summarizing the important results achieved by CDM-modified tissue engineering scaffolds in tissue organ reconstruction, helping readers to understand the developments in this field in recent years.

Molecular Insight into the Binding Property and Mechanism of Sulfamethoxazole to Extracellular Proteins of Anammox Sludge.

Antibiotics are widely found in nitrogen-containing wastewater, which may affect the operation stability of anaerobic ammonium oxidation (anammox)-based biological treatment systems. Extracellular polymeric substances (EPSs) of anammox sludge play a pivotal role in combining with antibiotics; however, the exact role and how the structure of the leading component of EPSs (i.e., extracellular proteins) changes under antibiotic stress remain to be elucidated. Here, the interaction between sulfamethoxazole and the extracellular proteins of anammox sludge was investigated via multiple spectra and molecular simulation. Results showed that sulfamethoxazole statically quenched the fluorescent components of EPSs, and the quenching constant of the aromatic proteins was the largest, with a value of 1.73 × 104 M-1. The overall binding was an enthalpy-driven process, with ΔH = -75.15 kJ mol-1, ΔS = -0.175 kJ mol-1 K-1, and ΔG = -21.10 kJ mol-1 at 35 °C. The O-P-O and C═O groups responded first under the disturbance of sulfamethoxazole. Excessive sulfamethoxazole (20 mg L-1) would decrease the ratio of α-helix/(ß-sheet + random coil) of extracellular proteins, resulting in a loose structure. Molecular docking and dynamic simulation revealed that extracellular proteins would provide abundant sites to bind with sulfamethoxazole, through hydrogen bond and Pi-Akyl hydrophobic interaction forces. Once sulfamethoxazole penetrates into the cell surface and combines with the transmembrane ammonium transport domain, it may inhibit the NH4+ transport. Our findings enhance the understanding on the interaction of extracellular proteins and sulfamethoxazole, which may be valuable for deciphering the response property of anammox sludge under the antibiotic stress.

Rapid quantitation of human epididymis protein 4 in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA).

A sensitive, rapid and homogeneous reaction measurement method for quantitation of human epididymis protein 4 (HE4) in human serum by amplified luminescent proximity homogeneous immunoassay (AlphaLISA) was described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one biotinylated monoclonal antibody combining on the surface of streptavidin coated donor beads, and "sandwiched" by another monoclonal antibody coated on acceptor beads. The coefficient variations of the method were lower than 10%, and the recoveries were in the range of 90-110% for serum samples. A value of 0.88pmol/l was identified as the minimum detectable dose of the present method for HE4. Compared with the results from electrochemiluminescence immunoassay kit (Roche) in 170 serum samples, there was a satisfied correlation coefficient of 0.984. The present assay demonstrated high sensitivity, wider effective detection range and excellent reproducibility for quantitation of HE4 can be useful for early screening and prognosis evaluation of patients with ovarian cancer.

Quantum Dot-Based Luminescent Oxygen Channeling Assay for Potential Application in Homogeneous Bioassays.

The unique photoproperties of quantum dots are promising for potential application in bioassays. In the present study, quantum dots were applied to a luminescent oxygen channeling assay. The reaction system developed in this study was based on interaction of biotin with streptavidin. Carboxyl-modified polystyrene microspheres doped with quantum dots were biotinylated and used as acceptors. Photosensitizer-doped carboxyl-modified polystyrene microspheres were conjugated with streptavidin and used as donors. The results indicated that the singlet oxygen that was released from the donor beads diffused into the acceptor beads. The acceptor beads were then exited via thioxene, and were subsequently fluoresced. To avoid generating false positives, a high concentration (0.01 mg/mL) of quantum dots is required for application in homogeneous immunoassays. Compared to a conventional luminescent oxygen channeling assay, this quantum dots-based technique requires less time, and would be easier to automate and miniaturize because it requires no washing to remove excess labels.

Rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (III) chelate microparticles-based lateral flow test strips.

Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (HT) and the control line (HC); the HT/HC ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0-1000 IU mL(-1)) for AFP with a low limit of detection (0.1 IU mL(-1)) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both <10%. Furthermore, in the analysis of human serum samples, excellent correlation (n = 284, r = 0.9860, p < 0.0001) was obtained between the proposed method and a commercially available CLIA kit. Results indicated that the Eu (III) chelate microparticles-based LFIA system provided a rapid, sensitive and reliable method for determining AFP in serum, indicating that it would be suitable for development in point-of-care testing.

Establishment of Magnetic Microparticles-Assisted Time-Resolved Fluoroimmunoassay for Determinating Biomarker Models in Human Serum.

In order to early screen and detect suspected biomarkers from pathogens and the human body itself, tracers or reaction strategies that can act as signal enhancers have been proposed forth at purpose. In this paper, we discussed the applicability of magnetic microparticles-assisted time-resolved fluoroimmunoassay (MMPs-TRFIA) for sensitive determination of potential analytes. Hepatitis B e antigen, antibody to hepatitis B surface antigen and free triiodothyronine were used as biomarker models to explore the reliability of the method. By coupling with bioprobes, MMPs were used as immunoassay carriers to capture target molecules. Under optimal condition, assay performance, including accuracy, precision and specificity, was outstanding and demonstrated satisfactory. To further evaluate the performance of the MMPs-TRFIA in patients, a total of 728 serum samples from hospital were analyzed for three biomarkers in parallel with the proposed method and chemiluminescence immunoassay kit commercially available. Fairly good agreements are obtained between the two methods via data analysis. Not only that but the reliability of MMPs-TRFIA has also been illustrated by three different reaction models. It is confirmed that the novel method modified with MMPs has been established and showed great potential applications in both biological detection and clinical diagnosis, including big molecule protein and low molecular weight haptens.

A magnetic nanoparticle-based time-resolved fluoroimmunoassay for determination of the cytokeratin 19 fragment in human serum.

A sensitive, rapid and novel measurement method for cytokeratin 19 fragment (CYFRA 21-1) in human serum by magnetic particle-based time-resolved fluoroimmunoassay (TRFIA) is described. Built on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating onto the surface of magnetic beads and "sandwiched" by another monoclonal antibody labeled with europium chelates. The coefficient variations of the method were lower than 7%, and the recoveries were in the range of 90-110% for serum samples. The lower limit of quantitation of the present method for CYFRA 21-1 was 0.78 ng/ml. The correlation coefficient of CYFRA 21-1 values obtained by our novel TRFIA and CLIA was 0.980. The present novel TRFIA demonstrated high sensitivity, wider effective detection range and excellent reproducibility for determination of CYFRA 21-1 can be useful for early screening and prognosis evaluation of patients with non-small cell lung cancer.

A time-resolved fluoroimmunoassay for the quantitation of rabies virus nucleoprotein in the rabies vaccine.

Sensitive, precise and rapid detection tests are needed in the quality control of rabies vaccine for rabies virus nucleoprotein. Previous studies for quantitation of rabies virus nucleoprotein focused on enzyme-linked immunosorbent assay (ELISA). A novel immunoassay for rapid determination of rabies virus nucleoprotein in rabies vaccine was first established by time-resolved fluoroimmunoassay (TRFIA). Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody coating in the wells and "sandwiched" by another monoclonal antibody labeled with europium chelates. The immunocomplex was retained after washing, and then adopted treatment with enhancement solution; fluorescence was then measured according to the number of europiumions dissociated. Levels of the rabies virus nucleoprotein were measured in a linear range (5-2500 mEU/mL) with a lower limit of quantitation (0.95 mEU/mL) under optimal conditions. The repeatability, recovery, and linearity of the immunoassay were demonstrated to be acceptable. The correlation coefficient of nucleoprotein values obtained by novel TRFIA method and ELISA method was 0.981. These results showed good correlation and confirmed that this sensitive, precise and rapid TRFIA was feasible and could be more suitable for the quality control in the process of rabies vaccine production than ELISA.

Development of an improved time-resolved fluoroimmunoassay for simultaneous quantification of C-peptide and insulin in human serum.

OBJECTIVES: Diabetes mellitus is a chronic disease affecting millions of people globally and resulting in significant death rates each year. A fast, inexpensive alternative to traditional testing and monitoring techniques is desirable, since secretion of insulin and C-peptide is impaired in diabetes mellitus. DESIGN AND METHODS: A highly sensitive immunoassay was developed for the simultaneous measurement of C-peptide and insulin levels in human serum, utilizing dual-label time-resolved fluoroimmunoassay (TRFIA) and magnetic particle technologies. This assay was characteristic for a single-step sandwich-type immunoassay, wherein antibody-coated magnetic particles were used as the solid phase and Eu(3+) and Sm(3+) chelate labels were used for detection. RESULTS: Antibody-coated magnetic particles in a TRFIA format performed well in addressing a number of quantitative needs. CONCLUSIONS: The results of this assay correlated well with commercial chemiluminescence assays and provided a number a advantages, including reduced sample volume, reduced reagent and personnel costs and reduced assay time, while maintaining the required clinical sensitivity.

A rapid and sensitive method based on magnetic beads for the detection of hepatitis B virus surface antigen in human serum.

Current clinically assays, such as enzyme-linked immunosorbent assay and chemiluminescence immunoassay, for hepatitis B surface antigen (HBsAg) are inferior in terms of either sensitivity and accuracy or rapid and high-throughput analysis. A novel assay based on magnetic beads and time-resolved fluoroimmunoassay was developed for the quantitative determination of HBsAg in human serum. HBsAg was captured using two types of anti-HBsAg monoclonal antibodies (B028, S015) immobilized on to magnetic beads and detected using europium-labeled anti-HBsAg polyclonal detection antibody. Finally, the assay yielded a high sensitivity (0.02 IU/mL) and a wide dynamic range (0.02-700 IU/mL) for HBsAg when performed under optimal conditions. Satisfactory accuracy, recovery and specificity were also demonstrated. The intra- and interassay coefficients of variation were 4.7-8.7% and 3.8-7.5%, respectively. The performance of this assay was further assessed against a well-established commercial chemiluminescence immunoassay kit with 399 clinical serum samples. It was revealed that the test results for the two methods were in good correlation (Y = 1.182X - 0.017, R = 0.989). In the current study, we demonstrated that this novel time-resolved fluoroimmunoassay could be used: as a highly sensitive, automated and high-throughput immunoassay for the diagnosis of acute or chronic hepatitis B virus infection; for the screening of blood or organ donors; and for the surveillance of persons at risk of acquiring or transmitting hepatitis B virus.

A novel immunoassay for the quantization of CYFRA 21-1 in human serum.

BACKGROUND: Cytokeratin 19 fragment antigen (CYFRA 21-1) is used to diagnose and monitor neoplasms. However, the main disadvantages of the currently available CYFRA 21-1 assays include heterogenous technology, being time-consuming, and having low through-put with low insensitivity. This study investigated the use of amplified luminescent proximity homogeneous immunoassay (AlphaLISA) for the quantization of CYFRA 21-1 in human serum. METHODS: The AlphaLISA kit was developed based on AlphaScreen detection technology with two different anti-CYFRA 21-1 monoclonal antibodies. One was coated on AlphaLISA acceptor beads and the other was biotinylated. Donor beads were coated with streptavidin. The test conditions were optimized and analytical performance was studied. RESULTS: The measurement range of AlphaLISA CYFRA 21-1 kit was 0.08-500 ng/ml. Assay detection limit was 0.08 ng/ml. The intra- and interassay coefficients of variation were 3.00-9.00% and 4.00-10.00%, respectively. There was no cross-reaction to alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 19-9 (CA19-9), cytokeratins 8 (CK8), and cytokeratins 18 (CK18). The correlation coefficient of blood samples involved was 0.974 between CYFRA 21-1-AlphaLISA assay and a commercial electrochemiluminescence immunoassay (ECLIA) CYFRA 21-1 kit (Roche). CONCLUSIONS: The AlphaLISA CYFRA 21-1 kit developed in this study had favorable performance characteristics for clinical application with acceptable analytical sensitivity, specificity, and accuracy.